PROJECT SUMMARY Pathogenic mycobacteria damage the phagosome and interact with the macrophage cytoplasm. The mycobac- terial factors that control bacteriolysis in the cytoplasm are unknown. The objective of this proposal is to define the genes controlling mycobacterial bacteriolysis in the macrophage cytoplasm. The central hypothesis for this application is that mycobacteria actively control bacteriolysis in the cytoplasm. To test this hypothesis, the fol- lowing specific aims will be tested. The applicant has adapted an established reporter for cytoplasmic bacteriol- ysis from Listeria monocytogenes for use with Mycobacterium. Under the first aim, the applicant proposes a targeted approach to define the impact of known virulence factors on mycobacterial bacteriolysis. The objective of Aim 1 is to define if known virulence pathways protect M. marinum from bacteriolysis. The applicant will test the working hypothesis that known virulence pathways that modulate the inflammasome protect Mycobacterium from bacteriolysis in the macrophage cytoplasm. M. marinum strains lacking specific virulence pathways will be generated and bacteriolysis will be measured during macrophage infection. Under the second aim, the applicant proposes an unbiased approach to identify genes required for cytoplasmic adaptation of mycobacterial patho- gens. The objective of Aim 2 is to Identify genes the control mycobacterial bacteriolysis. The applicant will test working hypothesis that several conserved pathways protect mycobacterial pathogens from bacteriolysis in the cytoplasm. Two complementary genetic screens to identify molecular pathways controlling mycobacterial bacte- riolysis in the cytoplasm during macrophage infection. The applicant expects that the successful completion of Aim 1 will determine if known virulence pathways protect mycobacteria from bacteriolysis. The completion of Aim 2 is expected to identify new pathways that promote mycobacterial survival in the cytoplasm. Completion of the proposed aims will contribute an initial understanding of the mechanisms required for preventing mycobac- terial bacteriolysis in the macrophage cytoplasm, moving the field in a new direction. This contribution will be significant because it will identify a new molecular mechanism underlying mycobacterial pathogenesis. The ap- plication is conceptually innovative because it focuses on mycobacterial determinants that protect mycobacteria from bacteriolysis in the cytoplasm, which represents a shift in focus for the field. The experimental design is innovative because it applies a novel indirect reporter of cytoplasmic bacteriolysis that has not previously been used to study mycobacterial species.

Project Narrative The proposed research is relevant to public health because the mycobacterial genes and pathways that promote interactions with the host during infection are potential targets for the development of new vaccines and therapeutics which would help to lessen tuberculosis. This research is relevant to NIH’s mission because defining mycobacterial virulence that promote survival during infection will contribute fundamental knowledge, which will be applicable to tuberculosis prevention, diagnosis and treatment.